Amonolyzed highly branched poly (beta-amino ester), preparation method thereof and application of ammonolyzed highly branched poly (beta-amino ester) in mRNA delivery

Abstract

The invention discloses ammonolysis highly branched poly (beta-amino ester) as well as a preparation method and application of the ammonolysis highly branched poly (beta-amino ester) in mRNA (messenger ribonucleic acid) delivery, and belongs to the technical field of biomedical materials. The ammonolysis highly branched poly (beta-amino ester) is obtained by terminating highly branched poly (beta-amino ester) through excessive amino monomers, the terminal of the ammonolysis highly branched poly (beta-amino ester) is provided with amide and hydroxyl groups, selective ammonolysis can be carried out under mild conditions in a precisely controllable manner, the toxicity of the material is reduced, and the stability of the material in serum is improved; hydrogen bonds in the structure can increase the interaction with mRNA and improve the compression capacity on nucleic acid; a main chain ester bond structure has good biodegradability, and an ammonolysis structure is beneficial to improvement of polymer mRNA encapsulation and release efficiency. In-vitro cell experiments prove that the ammonolysis highly branched poly (beta-amino ester) is low in toxicity and high in biocompatibility, can realize efficient mRNA delivery in mammalian cells, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a class of ammoniolyzed hyperbranched poly(β-amino ester), its preparation method, and its application in mRNA delivery. Background Art

[0002] Poly(β-amino ester) (PBAE) is a class of cationic degradable polymers prepared by the Michael addition reaction of acrylate and small molecule organic amines. Due to its unique pH responsiveness, biocompatibility, easy synthesis, adjustable chemical composition, topological structure, and functionality, it has attracted much attention in the fields of gene delivery, drug carriers, and biomaterials. Compared with linear poly(β-amino ester), hyperbranched poly(β-amino ester) has multiple terminal groups and a three-dimensional spherical topological structure, which has advantages in gene delivery.

[0003] Currently, hyperbranched poly(β-amino ester) is mainly synthesized by the traditional "bottom-up" strategy. By optimizing conditions such as monomer combination, topological structure, terminal groups, molecular weight, and distribution, the gene delivery efficiency is improved. However, the hyperbranched poly(β-amino ester) synthesized by the traditional "bottom-up" strategy mainly compresses mRNA through charge interaction. The excessive positive charge causes the complex nanoparticles formed after compressing mRNA to easily adsorb proteins in serum and aggregate, and it is not conducive to mRNA release. Moreover, a large number of ester bonds in the polymer backbone and a large number of amino groups at the terminals easily induce the formation of penetrating pores in the cell membrane through hydrophobic and electrostatic interactions, thereby generating cytotoxicity, which is not conducive to its clinical application. Summary of the Invention

[0004] In order to overcome the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a class of ammoniolyzed hyperbranched poly(β-amino ester), its preparation method, and its application in mRNA delivery, so as to solve the technical problem that the delivery efficiency and safety of the existing hyperbranched poly(β-amino ester) need to be improved.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, a class of ammoniolyzed hyperbranched poly(β-amino ester) is disclosed, and its structural formula is as follows:

[0007]

[0008] Among them, R1 is one of the following structures:

[0009]

[0010] R2 is one of the following structures:

[0011]

[0012] R3 is one of the following structures:

[0013]

[0014] R4 is one of the following structures:

[0015]

[0016] Wherein, m = 10 to 50.

[0017] In the second aspect of the present invention, a method for preparing the above-mentioned ammonolysis highly branched poly(β-amino ester) is disclosed, which includes the following steps:

[0018] Step 1: Prepare a highly branched poly(β-amino ester) with a double bond at the end by Michael addition reaction of acrylate monomers and small molecule amine monomers;

[0019] Step 2: Use an amino monomer as a capping agent to perform functional capping on the highly branched poly(β-amino ester) with a double bond at the end, and obtain an ammonolysis highly branched poly(β-amino ester) by controlling the concentration and reaction time of the amino monomer.

[0020] Preferably, in Step 1, the acrylate monomers include a diacrylate monomer and a triacrylate monomer with a molar ratio of (5 to 9):(1 to 5); or, the acrylate monomers include a diacrylate monomer and a tetraacrylate monomer with a molar ratio of (5 to 9):(1 to 5).

[0021] Further preferably, the structure of the diacrylate monomer is one of the following formulas:

[0022]

[0023] Preferably, the structural formula of the triacrylate monomer is The structural formula of the tetraacrylate monomer is

[0024] Preferably, in Step 1, the structure of the small molecule amine monomer is one of the following formulas:

[0025]

[0026] Preferably, in Step 1, the molar ratio of the acrylate monomer to the small molecule amine monomer is (1 to 3):1.

[0027] Preferably, in Step 1, the Michael addition reaction is carried out at 40 to 110 °C for 24 to 96 h.

[0028] Preferably, in step one, the acrylate monomer and the small molecule amine monomer are dissolved in dimethyl sulfoxide for the Michael addition reaction. In dimethyl sulfoxide, the total concentration of the acrylate monomer and the small molecule amine monomer is 200 - 500 mg / mL.

[0029] Preferably, in step two, the structure of the amino monomer is one of the following formulas:

[0030]

[0031] Preferably, in step two, the molar ratio of the capping agent to the acrylate monomer is (1 - 10):1.

[0032] Preferably, in step two, the capping reaction temperature is 25 - 50 °C, the reaction time is 24 - 96 h, and the concentration of the reaction solution during capping is 100 - 400 mg / mL.

[0033] In the third aspect of the present invention, the application of the above-mentioned ammonolysis highly branched poly(β - amino ester) in the preparation of an mRNA drug delivery carrier is disclosed.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] A class of ammonolysis highly branched poly(β - amino ester) provided by the present invention, compared with the highly branched poly(β - amino ester) synthesized by the traditional "bottom - up" strategy, the ammonolysis highly branched poly(β - amino ester) has amide bonds and hydroxyl groups at the ends, can be selectively ammonolyzed in a precisely controllable manner under mild conditions, can reduce the toxicity of the material and improve its stability in serum; the hydrogen bonds in the ammonolysis highly branched poly(β - amino ester) can increase the interaction with mRNA and improve the nucleic acid compression ability; the main chain ester bond structure in the ammonolysis highly branched poly(β - amino ester) has good biodegradability; the ammonolysis structure in the ammonolysis highly branched poly(β - amino ester) is beneficial to improving the polymer mRNA encapsulation and release efficiency. It is proved by in vitro cell experiments that the ammonolysis highly branched poly(β - amino ester) has low toxicity and high biocompatibility, and has a higher mRNA transfection efficiency compared with the un - ammonolyzed highly branched poly(β - amino ester) and the commercial product Lipfectamine MessengerMAX, and can achieve efficient mRNA delivery in mammalian cells, having good application prospects.

[0036] The preparation method of the above-mentioned ammoniolyzed highly branched poly(β-amino ester) provided by the present invention adopts a "top-down" synthesis strategy. Through the Michael addition reaction between acrylate monomers and small-molecule amine monomers, a highly branched poly(β-amino ester) with vinyl groups is prepared; then, effective capping and ammoniolysis are carried out through amino monomers, so as to obtain the end-functionalized ammoniolyzed highly branched poly(β-amino ester) in one step. This method has simple and efficient reactions, mild conditions, and low costs, and can rapidly synthesize this type of polymer with different molecular weights, structures, compositions, and properties. It has important application values, provides a new way for the modification of poly(β-amino ester) cationic polymers, and can enrich the types of this type of polymer. Description of the Drawings

[0037] Figure 1 It is the GPC spectrogram of the ammoniolyzed highly branched poly(β-amino ester) in Example 1;

[0038] Figure 2 It is the 1 1H NMR spectrogram of the ammoniolyzed highly branched poly(β-amino ester) in Example 1;

[0039] Figure 3 It is the microscopic morphology diagram of the complex formed by the ammoniolyzed highly branched poly(β-amino ester) and mRNA in Example 1 observed by Transmission Electron Microscope (TEM);

[0040] Figure 4 It is the fluorescence result diagram of cells after transfecting mRNA encoding green fluorescent protein in HeLa cells with the ammoniolyzed highly branched poly(β-amino ester) in Example 1;

[0041] Figure 5 It is the positive cell ratio result diagram after transfecting mRNA encoding green fluorescent protein in HeLa cells with the ammoniolyzed highly branched poly(β-amino ester), the non-ammoniolyzed highly branched poly(β-amino ester), and the commercial product Lipfectamine MessengerMAX (Thermo Fisher Scientific, LMrna001) in Example 1;

[0042] Figure 6 It is the GPC spectrogram of the ammoniolyzed highly branched poly(β-amino ester) in Example 2;

[0043] Figure 7 It is the 1 1H NMR spectrogram of the ammoniolyzed highly branched poly(β-amino ester) in Example 2;

[0044] Figure 8TEM micrograph of the complex formed by the ammoniolyzed hyperbranched poly(β - amino ester) and mRNA in Example 2;

[0045] Figure 9 Fluorescence result graph of cells after transfection of mRNA encoding green fluorescent protein by the ammoniolyzed hyperbranched poly(β - amino ester) in HeLa cells in Example 2;

[0046] Figure 10 Positive cell ratio result graph after transfection of mRNA encoding green fluorescent protein by the ammoniolyzed hyperbranched poly(β - amino ester), the non - ammoniolyzed hyperbranched poly(β - amino ester), and the commercial product Lipfectamine MessengerMAX (Thermo Fisher Scientific, LmRNA001) in HeLa cells in Example 2. Detailed implementation manners

[0047] To enable those skilled in the art to understand the features and effects of the present invention, the following provides only a general description and definition of the terms and phrases mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein shall have the ordinary meaning understood by those skilled in the art regarding the present invention. In case of conflict, the definition in this specification shall prevail.

[0048] The theories or mechanisms described and disclosed herein, whether right or wrong, shall in no way limit the scope of the present invention, that is, the content of the present invention can be implemented without being limited by any specific theory or mechanism.

[0049] In this article, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are only for the sake of brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be regarded as having covered and specifically disclosed all possible sub - ranges and individual values within the range (including integers and fractions).

[0050] In this article, unless otherwise specified, terms such as "comprising", "including", "containing", "having", or similar terms cover the meanings of "consisting of" and "consisting essentially of". For example, "A comprises a" covers the meanings of "A comprises a and others" and "A consists only of a".

[0051] In this article, for the sake of concise description, all possible combinations of all technical features in each embodiment or example are not described. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each embodiment or example can be combined arbitrarily, and all possible combinations should be considered as the scope described in this specification.

[0052] The present invention provides a class of ammonolysis highly branched poly(β - amino esters) with the following structural formula:

[0053]

[0054] Among them, R1 is one of the following structures:

[0055]

[0056] R2 is one of the following structures:

[0057]

[0058] R3 is one of the following structures:

[0059]

[0060] R4 is one of the following structures:

[0061]

[0062] In the formula, m = 10 - 50.

[0063] The present invention provides a preparation method of the above ammonolysis highly branched poly(β - amino esters), including the following steps:

[0064] Step 1, Michael addition reaction

[0065] Dissolve acrylate monomers and small - molecule amine monomers in dimethyl sulfoxide, react at 40 - 110 °C for 24 - 96 h, monitor the polymer molecular weight using gel permeation chromatography during the reaction, and terminate the reaction when the polymer molecular weight reaches 10000 - 100000 Da to obtain a highly branched poly(β - amino ester) with double - bonds at the ends;

[0066] Among them, the acrylate monomers include a diacrylate monomer and a triacrylate monomer with a molar ratio of (5 - 9):(1 - 5), or include a diacrylate monomer and a tetraacrylate monomer with a molar ratio of (5 - 9):(1 - 5); the molar ratio of the acrylate monomers to the small - molecule amine monomers is (1 - 3):1; in dimethyl sulfoxide, the total concentration of the acrylate monomers and the small - molecule amine monomers is 200 - 500 mg / mL; the small - molecule amine monomer is R2 - NH2 or R3 - NH2;

[0067] The diacrylate monomer is one of the following structures:

[0068]

[0069] The structure of the triacrylate monomer is The structure of the tetraacrylate monomer is

[0070] R2-NH2 is one of the following structures:

[0071]

[0072] R3-NH2 is one of the following structures

[0073]

[0074] Step 2. End-capping

[0075] Use an amino monomer to functionalize and end-cap the highly branched poly(β-amino ester) with a double bond at the end obtained in Step 1. React at 25 - 50 °C for 24 - 96 h, purify the product by precipitation method, and vacuum dry to obtain the ammonolyzed highly branched poly(β-amino ester);

[0076] Among them, the molar ratio of the end-capping agent to the acrylate monomer is (1 - 10):1, the concentration of the reaction solution during end-capping is 100 - 400 mg / mL, and the molecular weight of the ammonolyzed highly branched poly(β-amino ester) is controlled by controlling the concentration of the end-capping amino monomer and the reaction time. The amino monomer is R4-NH2, and the structure of R4-NH2 is one of the following structures:

[0077]

[0078] The present invention provides the application of the above ammonolyzed highly branched poly(β-amino ester) in mRNA delivery, and the steps are as follows:

[0079] Step 1. Cell culture

[0080] Culture HeLa cells under standard culture conditions, and then inoculate HeLa cells in a 96-well plate at a density of (1 - 2.0)×10 4 cells / well and culture for 24 h, then change the medium;

[0081] Step 2. Mix the ammonolyzed highly branched poly(β-amino ester) with the mRNA solution containing green fluorescent protein-encoding uniformly at a mass ratio of (5 - 200):1, let it stand and incubate for 20 - 30 min, and then transfer it to a 96-well plate for transfection. The mass of mRNA per well is 25 - 200 ng; after 24 h of transfection, observe and photograph the transfected cells under an inverted fluorescence microscope, and measure the proportion of green fluorescence-positive cells using a flow cytometer.

[0082] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0083] Conventional instrument equipment in the art is used in the following embodiments. For the experimental methods without specific conditions noted in the following embodiments, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Various raw materials are used in the following embodiments. Unless otherwise stated, commercially available products are used, and their specifications are the conventional specifications in the art. In the following embodiments, the preparation method of the unammonolyzed highly branched poly(β-amino ester) used in the control experiment was prepared with reference to Tailoring Highly Branched Poly(β-amino ester)s for Efficient and Organ-selective mRNA Delivery. Nano Letters, 2024, 24, 9368-9376.

[0084] Example 1

[0085] I. Preparation of ammonolyzed highly branched poly(β-amino ester)

[0086] 1. Acrylate monomers with a molar ratio of 1.1:1 (composed of bisphenol A ethoxylate diacrylate and pentaerythritol tetraacrylate with a molar ratio of 8:2) and small molecule amine monomers (composed of 2-aminoethylmorpholine and octylamine with a molar ratio of 7:3) were added to a flask containing dimethyl sulfoxide, so that the total concentration of acrylate monomers and small molecule amine monomers in dimethyl sulfoxide was 400 mg / mL. Then the flask containing the materials was placed at 90 °C for 30 h; the molecular weight of the polymer was monitored by gel permeation chromatography during the reaction. When the molecular weight of the polymer reached 10,000 - 100,000 Da, the reaction was terminated to obtain a highly branched poly(β-amino ester) with double bonds at the ends;

[0087] 2. 4-Morpholinopropylamine, which is 2 times the molar amount of the acrylate monomer used in step 1, was added to the flask in step 1, and the reaction was carried out at 25 °C for 48 h; the product was purified by precipitation method and dried in vacuum to obtain ammonolyzed highly branched poly(β-amino ester), and the structural formula is as follows:

[0088]

[0089] The GPC curve of the ammonolyzed highly branched poly(β-amino ester) is as Figure 1 shown. The molecular weight of this polymer is 21,000 Da, and the molecular weight distribution is 2.5. 1 The 1H NMR spectrum is asFigure 2 as shown

[0090] II. Delivery of Ammoniolyzed Hyperbranched Poly(β - amino ester) for mRNA

[0091] HeLa cells were seeded in a 96 - well plate at a density of 15,000 cells per well and cultured for 24 h, then the fresh medium was replaced. The ammoniolyzed hyperbranched poly(β - amino ester) prepared in Step I and mRNA ( EGFP mRNA, L - 7601, Trilink) were diluted in sodium citrate buffer solution at a mass ratio of 30:1, mixed evenly, and incubated statically for 25 min to form composite nanoparticles, and then transferred to a 96 - well plate for transfection. The dosage of mRNA per well was 50 ng. After 24 h of transfection, the transfected cells were observed and photographed under an inverted fluorescence microscope, and the proportion of green - fluorescence - positive cells was measured using a flow cytometer.

[0092] Meanwhile, un - ammoniolyzed hyperbranched poly(β - amino ester) and the commercial product Lipfectamine MessengerMAX (Thermo Fisher Scientific, LMRNA001) were used as controls respectively.

[0093] The microscopic morphology of the composite nanoparticles is as Figure 3 shown. The fluorescence microscope images of HeLa cells after transfection with ammoniolyzed hyperbranched poly(β - amino ester) are as Figure 4 shown. The results of the proportion of positive cells after transfection of ammoniolyzed hyperbranched poly(β - amino ester), un - ammoniolyzed hyperbranched poly(β - amino ester), and Lipfectamine MessengerMAX into HeLa cells are as Figure 5 shown.

[0094] Example 2

[0095] I. Preparation of Ammoniolyzed Hyperbranched Poly(β - amino ester)

[0096] 1. Acrylate monomers with a molar ratio of 1.5:1 (1,1'-isopropylidene bis(p - phenyleneoxy) di - 2 - propanol diacrylate and trimethylolpropane triacrylate with a molar ratio of 7:2) and small - molecule amine monomers (5 - amino - 1 - pentanol and n - octylamine with a molar ratio of 2:1) were added to a flask containing dimethyl sulfoxide, so that the total concentration of acrylate monomers and small - molecule amine monomers in dimethyl sulfoxide was 300 mg / mL. Then the flask containing the materials was placed at 90 °C for 36 h; during the reaction, gel permeation chromatography was used to monitor the molecular weight of the polymer. When the molecular weight of the polymer reached 10,000 - 100,000 Da, the reaction was terminated to obtain hyperbranched poly(β - amino ester) with double bonds at the ends;

[0097] 2. Add 1,11-diamino-3,6,9-trioxaundecane, which is 4 times the molar amount of the acrylate monomer, to the flask in Step 2, and react at 30 °C for 48 h; purify the product by precipitation method and dry it under vacuum to obtain the ammonolysis highly branched poly(β-amino ester), and the structural formula is as follows:

[0098]

[0099] The GPC curve of the ammonolysis highly branched poly(β-amino ester) is as Figure 6 shown. The molecular weight of this polymer is 21000 Da, and the molecular weight distribution is 2.5. 1 The 1H NMR spectrum is as Figure 7 shown.

[0100] II. Use the ammonolysis highly branched poly(β-amino ester) for mRNA delivery

[0101] Seed HeLa cells in a 96-well plate at a density of 20000 cells per well and culture for 24 h, then change to fresh medium. Dilute the ammonolysis highly branched poly(β-amino ester) prepared in Step 1 and mRNA in a mass ratio of 60:1 in sodium citrate buffer solution, mix evenly, and incubate statically for 30 min to form composite nanoparticles, and then transfer them to the 96-well plate for transfection, with the mRNA dosage of 25 ng per well. After 24 h of transfection, observe and photograph the transfected cells under an inverted fluorescence microscope, and measure the proportion of green fluorescence positive cells using a flow cytometer.

[0102] Meanwhile, use unammonolyzed highly branched poly(β-amino ester) and the commercial product Lipfectamine MessengerMAX (Thermo Fisher Scientific, LmRNA001) as controls respectively.

[0103] The microscopic morphology of the composite nanoparticles is as Figure 8 shown. The cell fluorescence microscope picture after the mRNA delivery efficiency of the ammonolysis highly branched poly(β-amino ester) transfection in HeLa cells is as Figure 9 shown. The cell fluorescence microscope pictures after the transfection of HeLa cells with ammonolysis highly branched poly(β-amino ester), unammonolyzed highly branched poly(β-amino ester), and Lipfectamine MessengerMAX are as Figure 10 shown.

[0104] The microscopic morphology results of the composite nanoparticles obtained from Example 1 and Example 2 ( Figure 3 and Figure 8) It can be seen that the composite nanoparticles are in a regular spherical structure, with good dispersibility and no agglomeration phenomenon. The particle size is about 150 nm, which is beneficial to endocytosis. The results of mRNA delivery efficiency of the ammoniated hyperbranched poly(β-amino ester) obtained from Example 1 and Example 2 in HeLa cells ( Figure 4 , Figure 5 , Figure 9 and Figure 10 ) It can be seen that almost all cells are green and the green fluorescence intensity is high. Compared with the unammoniated hyperbranched poly(β-amino ester) and the commercial product Lipfectamine MessengerMAX, the proportion of positive cells after transfection with the ammoniated hyperbranched poly(β-amino ester) is significantly higher, indicating that the ammoniated hyperbranched poly(β-amino ester) has very excellent mRNA transfection efficiency. In addition, after transfection, the cell morphology shows a plump and extended state, and no obvious cell debris is observed, indicating that the cell morphology is not affected after transfection, and the ammoniated hyperbranched poly(β-amino ester) has low cytotoxicity and good biocompatibility.

[0105] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A class of ammonolysis highly branched poly(β - amino ester), characterized in that, The structural formula is as follows: Among them, R1 is one of the following structures: R2 is one of the following structures: R3 is one of the following structures: R4 is one of the following structures: In the formula, m = 10 - 50.

2. The method for preparing the ammonolysis highly branched poly(β-amino ester) according to claim 1, characterized in that, It includes the following steps: Step 1: Prepare highly branched poly(β - amino ester) with double bonds at the ends by Michael addition reaction of acrylate monomers and small - molecule amine monomers. Step 2: Use amino monomers as capping agents to conduct functional capping on the highly branched poly(β - amino ester) with double bonds at the ends, and obtain ammonolysis - highly branched poly(β - amino ester) by controlling the concentration of amino monomers and the reaction time.

3. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 2, wherein In Step 1, the acrylate monomers include diacrylate monomers and triacrylate monomers with a molar ratio of (5 - 9):(1 - 5); or the acrylate monomers include diacrylate monomers and tetraacrylate monomers with a molar ratio of (5 - 9):(1 - 5).

4. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 3, wherein, The structure of the diacrylate monomer is one of the following formulas:

5. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 3, characterized in that, The structural formula of the triacrylate monomer is The structural formula of the tetraacrylate monomer is 6. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 2, wherein In Step 1, the structure of the small - molecule amine monomer is one of the following formulas:

7. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 2, characterized in that, In Step 1, the molar ratio of acrylate monomers to small - molecule amine monomers is (1 - 3):

1.

8. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 2, wherein In Step 2, the structure of the amino monomer is one of the following formulas:

9. The method for preparing an ammonolyzed highly branched poly(β-amino ester) according to claim 2, wherein In Step 2, the molar ratio of the capping agent to acrylate monomers is (1 - 10):

1.

10. Use of the ammonolysis - highly branched poly(β - amino ester) according to Claim 1 in the preparation of an mRNA drug delivery carrier.

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